Electrophoresis involves the separation of mixtures of molecules by differential migration of the molecules through a transport medium in an electric field. Many particles in an aqueous medium acquire an electrical charge due to ionization and thus move in response to an external electrical field. During electrophoresis a mixture of macromolecules is eventually separated into a series of distinct bands in order of charge density or size. Once the bands of molecules are separated they can be identified by suitable means such as staining and optical scanning. Electrophoresis in a gel medium is an important method of separating proteins, nucleic acids, and other macromolecules in mixture.
A given electric field may be altered by changing either or both of two parameters: (1) the voltage gradient, or intensity of the electric field; or (2) the direction, or orientation of the electric field. In conventional gel electrophoresis, at any given time a single electric field is applied to the gel, i.e., the intensity and orientation of the electric field being applied to the gel is constant in time throughout the electrophoretic separation.
In electrophoretic methods for separating large double stranded DNA molecules, several techniques have been advanced to increase the band resolution (i.e., increase the distance between bands without a corresponding increase in the width of the bands, or decrease the width of the bands without a corresponding decrease in the distance between bands). The advantages of pulsing the electric field (i.e., periodically changing the field orientation) during gel electrophoresis of high molecular weight double-stranded DNA was first demonstrated by Schwartz and Cantor. Schwartz et al., Cold Spring Harbor Symp. Quant. Biol. 47, 189 (1983); Schwartz and Cantor, Cell 37, 67 (1984); Cantor and Schwartz U.S. Pat. No. 4,473,452; Gardiner et al., Somatic Cell Mol. Genet., 12, 185 (1986).
A number of variants of pulsed-field gel electrophoresis (PFGE) have been described in the literature and are commercially available. In field-inversion gel electrophoresis (FIGE) the electric field alternates in polarity, and the durations of the "forward" and "back" pulses (the pulse amplitudes) are chosen to achieve a particular separation; net migration is achieved by using a longer time or higher voltage in one direction than in the other. U.S. Pat. No. 4,737,252; Carle et al., Science, 232, 65 (1986). In contour-clamped, homogeneous field electrophoresis (CHEF), the field direction is changed by 120.degree. while the field amplitude remains constant. Chu et al., Science 234, 1582 (1986). In rotating-gel electrophoresis, the field direction is changed by rotating the gel itself. Southern et al., Nucl. Acids. Res. 15, 5925 (1987). In transverse alternating field electrophoresis (TAFE), the field alternates in two directions approximately transverse to the plane of the gel. Gardiner et al., Somatic Cell and Molecular Genetics, 12, 185 (1986); U.S. Pat. No. 4,473,452. In programmable, autonomously controlled electrophoresis (PACE), the potentials of 24 electrodes are set independently, permitting exploration of a diversity of field directions and amplitudes. Birren et al., Nucl. Acids Res., 15, 7563 (1988). Pulsed fields have also been used to improve the separation of single-stranded DNAs. Birren et al., Nucl. Acids. Res. 18, 1481 (1990); Ulanovsky et al., Nature 343, 190 (1990).
Several variants of field inversion gel electrophoresis (FIGE) have been described. In their original description of FIGE, Carle et al. presented separation data for identical field amplitudes, E.sub.+ =E.sub.-, but different forward and back pulse durations, t.sub.+ .noteq.t.sub.- (where E.sub.+ indicates an electric field causing a molecule to move away from its starting point in a gel, E.sub.- indicates an electric field causing a molecule to move toward its starting point in a gel, t.sub.+ indicates the duration of a single pulse in field E.sub.+ and t.sub.- indicates the duration of a single pulse in field E.sub.-). Carle et al. noted that resolution in a particular size range could also be achieved if t.sub.+ =t.sub.- but E.sub.+ .noteq.E.sub.-. Carle et al., Science, 232, 65 (1986). Somewhat better separations are possible if different durations are used for t.sub.+ and t.sub.-, and different amplitudes are used for E.sub. + and E.sub.- ; this method has been termed Asymmetric Voltage Field-Inversion Gel Electrophoresis (AVFIGE). Birren et al., Nucl. Acids. Res. 18, 1481 (1990); Denko et al., Analyt. Biochem. 178, 172 (1989). A variant of AVFIGE, called Zero Integrated Field Electrophoresis (ZIFE) has been explored by Noolandi and Turmel. Turmel et al., in Electrophoresis of Large DNA Molecules, Birren and Lai (Eds.), Cold Spring Harbor Press, 101-132 (1990); Noolandi and Turmel, Pulsed Field Gel Electrophoresis, in Methods in Molecular Biology, vol. 12, p. 73, Burmeister and Ulanovsky (Eds.), Humana Press (1992). In ZIFE, both the pulse times and the pulse amplitudes are varied during a run, while in principle maintaining the product (E.sub.+ t.sub.+) equal to (E.sub.- t.sub.-). With this condition, .intg.Edt=0 over an integral number of cycles.
A common feature of pulsed-field gel electrophoresis (PFGE) and its variants is that the time-dependence is the same in all areas of the gel. At any given time a single set of parameters defines the electric field being applied to the gel, although those parameters may change during the course of the electrophoretic separation. In contrast, in MZPFGE, multiple distinct electric fields are created within the gel, with distinct spatial regions of the gel subjected to different fields at the same time.